Device and method for the determination of protein domain boundaries

ABSTRACT

A method and device to assist in the determination of protein domain boundaries is described. In particular the device is designed to provide a high throughput of proteolytic digestion of proteins to identify domains and their boundaries, for use in protein structure determination, in a manner that is amenable to automation. Proteases are immobilized in a convenient format such as a microtitre plate and preferably arranged in a matrix thereby allowing for simultaneous degradation of a protein by a number of proteases at a number of concentrations.

FIELD OF INVENTION

[0001] The present invention generally relates to methods fordetermination of protein structure and in particular concerns the use ofimmobilized proteases to identify domain boundaries of proteins, and anapparatus for digesting proteins.

BACKGROUND OF INVENTION

[0002] The molecular structure of proteins allows them play crucialroles in virtually all biological processes, including: enzymaticcatalysis, transport and storage; coordinated motion; mechanicalsupport; immune protection; generation and transmission of nerveimpulses; and control of growth and differentiation. In particular, theside chains of the different amino acids that comprise proteins, enablesthese long macromolecules to fold into distinctive structures and formcomplementary surfaces and clefts and enables them to specificallyrecognize and interact with highly diverse molecules. The catalyticpower of enzymes comes from their capacity to bind substrates in preciseorientations and to stabilize transition states in the making andbreaking of chemical bonds. Conformational changes transmitted betweendistant sites in protein molecules are at the heart of the capacity ofproteins to transduce energy and information. Thus, the threedimensional structure of a protein is the key to its ability to functionin virtually all biological processes.

[0003] Discussions pertaining to protein architecture concern fourlevels of structure which are described as follows. Primary structure isthe amino acid sequence. Secondary structure refers to the spatialarrangement of amino acid residues that are near one another in thelinear sequence. Some of these steric relationships are of a regularkind, giving rise to a periodic structure. Tertiary structure refers tothe spatial arrangement of amino acid residues that are far apart in thelinear sequence, and to the pattern of disulfide bonds. The term,quaternary structure, refers to proteins containing more than onepolypeptide chain where each polypeptide chain is referred to as asubunit, and the quaternary structure refers to the spatial arrangementsubunits and the nature of their contacts.

[0004] Some polypeptide chains fold into two or more compact regionsthat may be joined by a flexible segment of polypeptide chain. Thesecompact globular units, called domains, range in size from about 50 to400 amino acid residues, and they seem to be the modular units fromwhich proteins are constructed. While small proteins may contain only asingle domain, larger proteins contain a number of domains, which areoften connected by relatively open lengths of polypeptide chain.Although all information required for folding a protein chain iscontained in the protein's amino acid sequence, it is not yet known howto “read” this information so as to predict the detailedthree-dimensional structure of a protein whose sequence is known.Consequently, folded conformation currently can only be determined by anelaborate X-ray diffraction analysis performed on crystals of theprotein or, if the protein is very small, by nuclear magnetic resonance(“NMR”) techniques. Although considerable advances are being made in thearea of high field NMR, presently, the only method capable of producinga highly accurate three dimensional structure of most proteins is by theapplication of X-ray crystallography.

[0005] Recent advances in the field of X-ray crystallography, such ashigh speed computer graphics and X-ray area detection technologies, haverevolutionized the pace at which three-dimensional structures can bedetermined. The resulting three dimensional structure produced from theprotein crystals can have enormous implications in the fundamentalunderstanding of molecular biology such as how enzymes perform variouscatalytic activities, switch on biological pathways, or transportmolecules within the circulatory system. In the past few years thedetermination of protein structures important as therapeutic targets hasmade possible the rational design of new, more effectivepharmaceuticals.

[0006] The technique of X-ray crystallography utilizes the diffractionof X-rays from crystals in order to determine the precise arrangement ofatoms within the crystal. The limiting step in the technique involvesthe growth of a suitable crystalline sample. This requires the growth ofreasonably ordered protein crystals (crystals which diffract X-rays toat least 3.0 angstroms resolution or less).

[0007] Because of the complexity of proteins, obtaining suitablecrystals can be quite difficult. Typically, several hundred to severalthousand individual experiments must be performed to determinecrystallization conditions, each examining a matrix of pH, buffer type,precipitant type, protein concentration, temperature, etc. This processis extremely time consuming and labour intensive.

[0008] A strategic approach has been developed to identify proteindomains that are amenable to NMR analysis or X-ray crystallography.Different domains of a protein may be linked together by interveningsections of polypeptide chain to form the protein molecule. Analysis ofa single domain is more easily conducted in isolation from its parentprotein. The determination of an individual domain structure facilitateselucidation of the parent structure. Limited proteolysis has been usedto isolate and identify stable domains of proteins, which have a highlikelihood of being good targets for protein structure determination.The approach is based upon the observation that low concentrations ofone or more specifically chosen proteases cleave proteins intoproteolytically stable domains amenable to NMR analysis orcrystallography (Morin, P. E; et aL Proc. Nad. Acad. Sci. 1996, 93,10604-10608; Barswell, J. A.; et al. J. Bio. Chem. 1995, 270,20556-20559; Pfuetzner, R. A; et al. J. Bio. Chem. 1997, 272, 430-434;and Malhotra, A; el aL Cell 1996, 87, 127-136).

[0009] Limited proteolysis has been conducted to isolate and identifystable domains of proteins. Generally, according to this process, theprotein is incubated with four to six different proteases at differentconcentrations for different amounts of time. Typical protease digestionreactions are conducted by dissolving an enzyme in water therebyallowing the enzyme to act on a substrate in an aqueous solution.However, the fact that the enzyme reaction is a homogeneous reaction inan aqueous solution is a great hindrance to performance of a continuousreaction in industrial applications and also makes it very difficult torecover remaining active enzymes for repeated use after the reaction. Inaddition, complicated operational procedures are necessary forseparation and purification of the reaction product.

[0010] The digestion products can be analyzed by SDS/polyacrylamide gelelectrophoresis and proteolytically stable fragments can be identifiedon the basis of approximate mass. These products can then be isolated byreverse phase chromatography and an accurate mass determination can beperformed by mass spectrometry. The accurate mass of a proteolyticfragment is sufficient to uniquely identify the boundaries of thefragment within a sequence of a protein. The identification of theproteolytic fragment sequence facilitates the recombinant preparation ofthe domain in sufficient quantities for X-ray and NMR analysis.

[0011] A limitation of the aforementioned strategy is the time consumingnature of cleaving, identifying and isolating the domains of a proteinfrom the digestion solution.

SUMMARY OF THE INVENTION

[0012] The present invention to provides a method and device to assistin the determination of protein domain boundaries. In accordance with anaspect of the invention there is provided a method for the preparationof proteolytically digested fragments of a protein in one step forpurification and further processing for determination of domains andtheir boundaries in the protein, The method developed by presentinventor comprises a one step degradation of protein which comprisescontacting a quantity of the protein with two or more concentrations ofone or more immobilized proteases for a time sufficient to allowdegradation of the protein to provide digested fragments and thenseparating the immobilized protease from the fragments. Each protease isimmobilized in a compartment in an apparatus which comprises a pluralityof compartments, each compartment containing a quantity of a proteaseimmobilized on a surface in each compartment. According to a preferredembodiment each compartment contains a concentration of the proteasedistinct from the concentration of the protease in every othercompartment. Preferrably the surface upon which the protease isimmobilized has been treated with a blocker of surface interaction. Theprotein is contacted with the protease in each compartment at about thesame time.

[0013] In accordance with a further aspect of the invention the methodis automated allowing for simultaneous addition of a quantity of theprotein to each of the compartments. Downstream automation removesprotolytically treated fragments to a purification step to yield one ormore samples for further processing and structure determination.Accordingly, the present invention provides a high throughput method anddevice for enzymatically cleaving proteins into domains which allows foran efficient means to identify protein domain boundaries for use inprotein crystallization, in a manner that is amenable to automation.

[0014] In accordance with one aspect of the present invention a deviceof the invention is connected downstream to an automatic means of addinga protein solution, or a solution of a protein fragment, and upstream toan automatic means of removing and subjecting the proteolyticallydigested product to a purification step.

[0015] In accordance with another aspect of the present invention thereis provided a method for determining the boundaries of a proteolyticallydigested fragment of a protein which comprises the steps: (i) incubatinga protein, or a fragment thereof, with at least one immobilized proteaseto yield protein fragments; and (ii) subjecting the resulting proteinfragments to one or more purification steps to isolate the fragment(s)of interest; (iii) subjecting the isolated fragment(s) to eithernanospray or matrixassisted time-of-flight mass spectrometry; (iv)matching the mass of the proteolytic fragment to the protein sequence ofthe protein originally digested.

[0016] According to another aspect of the present invention there isprovided an apparatus for degradation of a protein, where the apparatuscomprises a plurality of compartments, with at least two differentconcentrations of protease in separate compartments. In a preferredembodiment each compartment contains a concentration of proteasedistinct from the concentration of protease in every other compartment.

[0017] The present invention also includes a kit containing the deviceof the present invention together with appropriate reagents andinstructions for its use.

[0018] Once proteins domains have been determined by the methods of thepresent invention, these protein domains can be used in screens ofprotein-protein interaction, such as for example, by affinitychromatography.

[0019] Advantages of the present invention include the primary factorthat proteases and protease fragments do not substantially contaminatethe proteolytically digested protein fragments as the proteases areimmobilized and not in solution.

[0020] Another advantage is the ability to re-use the treated plates.Another advantage is the reproducibility and as mentioned, highthroughput of protease digestion.

[0021] Because many devices can be generated at the same time, there isan efficiency of production and consistency of immobilized proteaseconcentration that can be attained by this invention that is notcurrently available in the art. Some of the plates with an immobilizedprotease can be mass produced and stored for at least one week, at 4°C., while maintaining activity. Plates can be manufactured as standardsor as custom models as requested.

[0022] In contrast to autolytic proteases (and mixtures of proteasesthat can degrade due to one protease cleaving another protease, insolution), the ready use format of the present invention provides animmobilized product that: (1) is essentially unable to undergo autolyticcleavage; and (2) is essentially unable to degrade due to one proteasecleaving another protease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 shows a comparison of PAGE results of a limited digestionof the transcription factor TFIIS from the yeast, Saccharomycescerevisiae. in solution and in microtitre plates.

[0024]FIG. 2 shows the PAGE results of samples of limited digestion ofvarious proteins in microtitre plates by trypsin and chymotrypsin whereA represents the results with RNA polymerase B5; B is RNA polymerase B6;C is RNA polymerase B8; and D is a bacterial protein parB.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Current limitations in the art of identifying stable domains ofproteins for use in structure determination are overcome by use of thisinvention which provides a device and method for conducting limitedproteolysis of proteins to identify protein domains useful for structuredetermination. In particular the device is designed to provide a highthroughput of proteolytic digestion of proteins to identify domains andtheir boundaries, for use in protein structure determination, in amanner that is amenable to automation.

[0026] As used herein the word “protein” includes the terms “peptide”and “polypeptide” and includes a fragment of a protein as well as anamino acid chain which provides a fully constituted molecular structuresuch as the transcription factor TFIIS from the yeast, Saccharomycescerevise.

[0027] As used herein, the phrase “proteolytically digested protein”means the degradation of a protein by an enzyme or protease and includeslimited degradation of a protein by an enzyme or protease.

[0028] As used herein the phrase “solid support” includes any substancewhich is capable of interacting with a protease or other enzyme andholding it out of solution, and includes for examples organic andinorganic support materials as described herein.

[0029] As used herein “compartment” includes any structure which iscapable of supporting an immobilized protease, for example, a well in amicrotitre plate, a column containing a resin or other bead-likeparticle. It will be understood that there may be duplicates andtriplicates of some concentrations within a plurality of compartments ofan apparatus of the invention, so long as over all there are at leasttwo or three different concentrations. Further, it is understood that anumber of apparatuses of the invention may be used in parallel to createa super matrix with numerous repeats of a particular concentration allwithin one apparatus with each apparatus having differing concentrationsof protease.

[0030] Method of the Invention

[0031] The present invention provides a method for the preparation ofproteolytically digested fragments of a protein, free of protease, forthe determination of domains and the boundaries of the domains in saidprotein, the method comprises:

[0032] (1) contacting a quantity of the protein with at least twoconcentrations of a protease under conditions which result in digestedfragments of the protein, wherein each of the concentrations of proteaseis immobilized in a separate compartment; and

[0033] (2) separating said fragments from the immobilized protease.Preferably the compartment is in an apparatus which comprises aplurality of compartments.

[0034] According to one embodiment of the method compartment containinga quantity of the protease contains a concentration of the proteasedifferent from the concentration of the protease in every othercompartment. Preferably at least three concentrations of protease are inat least three different compartments.

[0035] According to another embodiment the plurality of compartmentsforms a matrix in a linear format with at least three increasingconcentrations of protease in the compartments. Preferably the matrixcontains more than one protease.

[0036] According to a preferred embodiment of the method thecompartments form a matrix in a two-dimensional format wherein: (i)distinct concentrations of each protease are aligned in increasingconcentration, along a first axis or concentration axis; and (ii)different proteases are aligned along a second axis that isperpendicular to the first axis.

[0037] A protease for use in ta method of the invention is selected fromthe group consisting of aminopeptidase M; bromelain; carboxypeptidase A,B and Y; chymopapain; chymotrypsin; clostripain; collagenase; elastase;endoproteinase Arg-C, Glu-C and LysC; Factor Xa; ficin; Gelatinase;kallikrein; metalloendopeptinidase; papain; pepsin; plasmin;plasminogen; peptidase; pronase; proteinase A; proteinase K; subsilisin;thermolysin; thrombin; and trypsin. Preferably the proteases aretrypsin, chymotrypsin, proteinase K or papain.

[0038] According to a preferred embodiment the concentrations of each ofthe proteases along the first axis in each case is from about 50 μg/mLto about 0.5 μg/mL. More preferably the concentration for each of theproteases is about 50 μg/mL in one container of a concentration axis;about 25 μg/mL in a second container of the concentration axis; about 5μg/mL in a third container of the concentration axis; about 2.5 μg/mL ina fourth container of the concentration axis; about 0.5 μg/mL in a fifthcontainer of the concentration axis; about 0 μg/mL in a sixth containerof the concentration axis.

[0039] A preferred embodiment of the method requires that the surfaceupon which the protease is immobilized has been treated with a blockerof surface interaction. Preferably the blocker is selected from thegroup consisting of BSA and beta-octyl glucoside.

[0040] According to another aspect of the method the surface to whichthe protease(s) is/are immobilized is selected from the group consistingof an organic support of material selected from the group consisting ofpolyesters, polyamides, polyacrylates, polymethacrylates,polyacrylamides, poly(acrylic acid)m, poly(methacrylic acid);poly(galacturonic acid); poly(aspartic acid); ethylene-maleic anhydridecopolymers; polyolefins; cellulose; cellulose derivatives; agarose gels;dextran gels and derivatives thereof, polysaccharides; polypeptides;collagen; and an inorganic support material selected from the groupconsisting of siliceous and nonsiliceous metal oxides.

[0041] In a preferred embodiment of the method the apparatus is amicrotitre plate and the time for incubation of protein with immobilizedprotease is between about 2 to 4 hours. Preferably the temperature forthe reaction between immobilized protease and protein is roomtemperature. According one embodiment of the method only limiteddigestion of the protein occurs.

[0042] Accordingly carrying out the method of the present inventionprovides proteolytically digested protein fragments substantially freeof digesting protease by using proteases that are immobilized on a solidsupport such as a microtiter well. Coupling of the immobilized proteaseplates to a chromatographic step for separation of the protein fragmentsthereby generated, prepares the fragments for further analysis such assequence determination or mass spectrometry. The design of the deviceallows for a high throughput of protein samples in addition to beingparticularly suitable for automation. As such the present inventionprovides a method for determining the boundaries of a proteolyticallydigested fragment of a protein which comprises the steps: (i) incubatinga protein, according to the methods described herein to yield proteinfragments; (ii) isolatation of fragments) of interest; (iii) determiningthe mass of the isolated fragment(s); and (iv) matching the mass of theproteolytic fragment(s) to a protein amino acid sequence of the proteindigested. Preferably the determination of mass is by a method selectedfrom the group consisting of nanospray time-of-flight mass spectrometryand matrix-assisted time-of-flight mass spectrometry. Also, preferablythe protein fragment isolation is carried out by a technique selectedfrom the group consisting of High Performance Liquid Chromatograph(HPLC) and sodium dodecylsulphate (SDS) polyacrylamide gelelectrophoresis (PAGE). The loading and transference of the proteinsamples to the device and, following the reaction, to a detector such asHPLC or mass spectrometer, can be performed by automated means known inthe art. According to a preferred embodiment the method is automated.

[0043] Automated sampling and automated transfer and mixing of solutionsand solvents is routine. Many commercial automated apparatuses areavailable and save considerable hours and days of sample manipulation.For example, auto samplers for HPLC, gas chromatography and massspectrometry are all commercially available. In addition, commercialsolution handling devices are readily customised to suit the usersneeds. The employment of such automated handlers in conjunction withprotease immobilized plates removes the steps of manipulating a proteasesolution and subsequent removal of unwanted protease reagent andprotease fragments.

[0044] The use of the protease plates allows highly parallel domainmapping. The plates, with commercially available robotics, can becoupled with HPLC and mass spectrometry to make an integrated system forthe rapid molecular identification of protein domains.

[0045] Apparatus of the Invention

[0046] An apparatus of the present invention can be used to treat aprotein solution containing whole proteins or fragments thereof tolimited proteolysis to generate stable domains of the proteins forfurther analysis. Accordingly the present invention provides anapparatus for degradation of a protein, where the apparatus comprises aplurality of compartments, each compartment containing a quantity of aprotease immobilized on a surface in each compartment wherein at leasttwo compartments contain different concentrations of the protease.Preferrably each of the compartments of the apparatus contain aconcentration of the protease different from the concentration of theprotease in every other compartment.

[0047] According to a preferred embodiment an apparatus according to thepresent invention contains at least three concentrations of protease inthree compartments. Preferably a plurality of compartments forms amatrix in a linear format with at least three increasing concentrationsof protease in at least three of the compartments. More preferably theapparatus contains more than one protease.

[0048] According to another embodiment of an apparatus of the presentinvention a plurality of compartments forms a matrix in atwo-dimensional format wherein: (i) distinct concentrations of eachprotease are aligned in increasing concentration, along a first axis orconcentration axis; and (ii) different proteases are aligned along asecond axis that is perpendicular to the first axis.

[0049] According to a preferred embodiment an apparatus according to thepresent invention is a microtitre plate. A “microtitre plate” asreferred to herein is well known to those skilled in the art and is asample plate having one or more wells for receipt of a sample, themicrotitre plate being suitable for use in an automated process. A platewell within a single microtitre plate can contain one or more proteasesat a single concentration or differing concentrations or be a controlblank. Due to the open layout of the microtiter wells, and the matrix ofproteolytic enzymes, the design of the device permits a high throughputlevel of protein treatment that significantly diminishes the timerequired for an equivalent digestion procedure that is familiar in theart. Typically, such plates contain 96 wells, but higher volume platessuch as 380 or more are also contemplated to function in the presentinvention.

[0050] On such microtitre plates a protease digestion matrix isgenerated with different proteases along one axis and increasingconcentrations along the other axis.

[0051] Different proteases can be used, both alkaline and acidic.Moreover, given the existence of a multitude of known proteases and theapplication of recombinant DNA technology to the study and production ofprotease analogs, the art has yet to develop completely. Many proteasesare available and can be used in this device and procedure, for example:aminopeptidase M; bromelain; carboxypeptidase A, B and Y; chymopapain;chymotrypsin; clostripain; collagenase; elastase; endoproteinase Arg-C,Glu-C and LysC; Factor Xa; ficin; Gelatinase; kallikrein;metalloendopeptinidase; papain; pepsin; plasmin; plasminogen; peptidase;pronase; proteinase A; proteinase K; subsilisin; thermolysin; thrombin;and trypsin. Preferably the proteases are trypsin, chymotrypsin,proteinase K or papain.

[0052] The larger the number of proteases with different specificity,the greater the procedural flexibility provided by one or moremicrotitre plates. In a preferred embodiment a single plate has six ormore different immobilized proteases. Each protease is dispensed along asingle row, with the concentration in the first well at 50 microgramsper millilitre and each subsequent well with a two-fold dilution.According to a preferred embodiment an apparatus according to thepresent invention has concentrations of each of the proteases along thefirst axis from about 50 μg/mL to about 0.5 μg/mL. More preferably theconcentration for each of the proteases is about 50 μg/mL in onecontainer of a concentration axis; about 25 μg/mL in a second containerof the concentration axis; about 5 μg/mL in a third container of theconcentration axis; about 2.5 μg/mL in a fourth container of theconcentration axis; about 0.5 μg/mL in a fifth container of theconcentration axis; about 0 μg/mL in a sixth container of theconcentration axis. Preferably the surface upon which the protease(s) isare immobilized has been treated with a blocker of surface interactionwhere the blocker is selected from the group consisting of BSA andbeta-octyl glucoside.

[0053] An apparatus of the invention can be stored for a period of atleast a week, at 4° C. Preferably before storage the apparatus islyophilized.

[0054] Immobilization of the enzyme on the solid support is readilyaccomplished by various methods which are known. Various methods areknown for immobilization of enzymes (refer to, for example, O. R.Zaborsky, “Immobilized Enzymes”, C.R.C. Press, 1973; or “ImmobilizedEnzymes”, edited by Ichiro Chihata, Kodansha, 1975; see also see I.Chibata, Editor, “Immobilized Enzymes”, Halsted Press, John Wiley &Sons, Inc., New York, 1978, pp. 1-73). They can roughly be classifiedinto the following four groups: (1) physical or ionic adsorption method;(2) covalent attachment method; (3) entrapment method; and (4)crosslking method.

[0055] In a preferred embodiment, the device is generated byimmobilizing the proteases by overnight incubation at room temperature.

[0056] The solid support generally can be either organic or inorganic.Examples of organic supports include, among others, polyesters, such aspoly(ethylene terephthalate); polyamides, such as nylon 6 and nylon 6.6;polyacrylates; polymethacrylates; polyacrylamides; poly(acrylic acid);poly(methacrylic acid); poly(galacturonic acid); poly(aspartic acid);ethylene-maleic anhydride copolymers; polyolefins, such as polyethylene,polypropylene, polybutene, and polybutadiene; polystyrene;poly(aminostyrene); poly(vinyl chloride); poly(vinyl alcohol);poly(vinylidene chloride); cellulose and derivatives thereof; agarosegels; dextran gels and derivatives thereof, polysaccharides;polypeptides; collagen; and the like.

[0057] The inorganic supports can be classified as siliceous ornonsiliceous metal oxides. Examples of siliceous supports include, amongothers, glass, silica, wollastonite, bentonite, cordierite, and thelike. Examples of nonsiliceous metal oxides include, among others,alumina, spinel, apatite, nickel oxide, titania, zirconia, and the like.

[0058] In general, the support can be in any desired shape or form thatis a continuous, shaped article such as a flat or curved sheet or athree-dimensional article such as a rectangular or cylindrical tube. Asa practical matter, however, the support most often will be a microtiterplate or a similar shaped support.

[0059] For examples of procedures for immobilizing enzymes on inorganicsupports, by way of illustration only, see U.S. Pat. No. 3,519,538(which corresponds with French Patent No. 2,020,527), U.S. Pat. No.3,556,945 (which corresponds with French Patent No. 2,001,336), and U.S.Pat. Nos. 3,666,627 and 3,802,997 (which correspond with French PatentNo. 2,020,661).

[0060] In a preferred embodiment, the non-specific binding sites on themicrotitre wells are blocked for a period of time (three hours) with anappropriate blocking solution containing 0.1% betaoctylglucoside,although depending upon the support material chosen other blockers willbe preferred, such as for example bovine serum albumin (BSA).

[0061] Kit

[0062] In another embodiment, the present invention relates to a kit forconducting limited proteolysis digestion of proteins or proteinfragments for structure determination analysis comprising at least onecontainer including the above-described device. The proteolysis devicecan be presented in a commercially packaged form, a packaged combinationof one or more containers, devices, or the like, holding the necessaryreagents and usually including written instructions describing theperformance of the digestion procedure. Reagent systems of the presentinvention involve all possible configurations and compositions forperforming the various digestion formats described herein.

[0063] In a preferred embodiment, the kit further comprises othercontainers comprising one or more of the following: wash reagents,dilution reagents, proteolysis stopping reagents and control proteinswith known digestion patterns and instructions for use of the kit.

[0064] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separated containers. Such containers includeglass or plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment or another. Suchcontainers will include a container which will accept the microtiterplate, a container which contains wash reagents and proteolysis stoppingreagents (eg. acetic acid).

[0065] In an alternative embodiment, the kit will include the materialsto prepare a device of the present invention, which would therefore,include proteolytic enzymes, blocking reagents and buffer materials. Oneskilled in the art will readily recognize that the proteolysis device ofthe present invention can readily be incorporated into one of theestablished kit formats which are well known in the art.

[0066] The following non-limiting examples are illustrative of thepresent invention:

EXAMPLES Example 1

[0067] General Experimental Protocol for Immobilizing Proteases onMicrotiter Plates

[0068] In this example, four proteolytic enzymes are immobilized on amicrotiter plate.

[0069] Reagents

[0070] The reagents were prepared as follows. A buffer solution of TBS,50 mM Tris pH 8.0, 150 mM NaCl is prepared for use with the remainingsolutions. A solution of TBS, 0.01% beta-octa-glucoside is prepared as ablocking buffer. The following proteases are prepared: Chymotrypsin, 0.5mg/ml (in TBS); Trypsin, 0.5 mg/ml (in TBS); Papain, 0.5 mg/ml (in TBS);and Proteinase K, 0.5 mg/ml (in TBS). The protein to be digested isprepared at 65 μg/ml (diluted in TBS).

[0071] Preparation of Immobilized Protease Plate

[0072] In this example of device preparation, Nunclon surface 96 wellmicrotiter plates are used. The proteases are immobilized onto theplates by aliquotting 45 μl TBS into each well. Six wells are used foreach protease to generate a series for each protease, each seriesoperating at decreasing concentrations across the wells by adding 5 μlof protease stock solution to the first well, 2.5 μl to the second well,etc. The serial dilution of the wells yield final concentrations ofproteases of 50 μg/ml, 25 μg/ml, 5 μg/ml, 2.5 μg/ml, and 0.5 μg/ml. Thelast well is left for protein only. The plate is covered and placed in asealable bag with a wet paper towel. The resulting plate is incubatedovernight at 4° C.

[0073] Blocking of Non-Specific Sites on Microtiter Plate

[0074] The protease solution is removed from the wells by flicking theplate a few times while inverted. The wells are washed once withblocking buffer (100 μl) and the excess solution is removed. 100 μl ofblocking buffer is added and incubate for 30 minutes at 4° C. Theblocking buffer is removed and the wells are washed once (100 μl) withTBS and the excess TBS is removed. The plates are now ready for use orcan be lyophilized and stored at −20° C. until needed.

[0075] Digestion of an Protein by an Immobilized Protease

[0076] 30 μl of the protein solution of interest is added to each well.The resulting plate is then incubated at room temperature for 2-4 hours.

[0077] Analysis of Proteolytically Digested Protein

[0078] If the protein fragments are to be separated and analyzed by MassSpectrometry, it is necessary to remove a sample of a proteolyticallydigested protein (5-10 μL) and stop the proteolysis by adding aceticacid to the sample until a final concentration of 1% is reached.

[0079] If the protein fragments are to be separated and analyzed by SDSPAGE Gel Electrophoresis, the proteolysis of protein is stopped byadding 5×protein loading dye to each well. The sample in each well isheated to approximately 90° C. for approximately 5 minutes. 20-25 μl ofthe sample is loaded onto a SDS PAGE gradient gel (5-18%), and run underelectrophoresis followed by a Commassie stain to visualize the proteinfragments.

Example II

[0080] Comparison of Immobilized Protease Digestion to Solution ProteaseDigestion

[0081] The protease plates were used to digest five different proteinswhose proteolytic digestion pattern in solution was already known. Thepattern of digestion for the immobilized proteases mimicked the solutiondigestion pattern. Thus, we have shown that the immobilized proteasesplates could be used to identify stable domains of several differentproteins. Shown in FIG. 1 is an example of a limited digestion of thetranscription factor TFIIS from the yeast, Saccharomyces cerevisiae. Thefive lanes shown correspond to the concentration of proteases used (asdiscussed above in Example I). FIG. 2 illustrates results of digestionon immobilized protease plates of RNA polymerase B5 (see A); RNApolymerase B6 (B); RNA polymerase B8 (C); and D is a bacterial proteinparB. Each of these four proteins were digestd by trypsin (panels on theleft) and chymotrypsin (panels on the right). These digests comparefavourably with similar digests in solution (data not shown).

Example 111

[0082] Limited Proteolysis

[0083] Four different proteases, trypsin, chymotrypsin, papain andproteinase K (Sigma) were immobilized on plastic 96-well microtitreplates (Nuclon) in the following manner. The protease stocks were made0.5 mg/ml in TBS (50 mM Tris pH 8, 150 mM NaCl). A serial dilution ofeach protease was prepared to final concentrations of 50 μg/ml, 25μg/ml, 5 μg/ml, 2.5 μg/ml and 0.5 μg/ml in TBS. 50 μl of each dilutionwas applied to different wells in a row of the microtitre plate. Theplate with the arrayed protease dilutions was then incubated overnightat 4° C. in a sealed bag containing a wet paper towel.

[0084] The protease solution was then removed and the wells washed with100 μl of blocking buffer (TBS, 0.01% beta-octyl glucoside). The firstwash was discarded and the non-specific binding sites on the microtitrewells were blocked with an additional 30 minute incubation at 4° C. withan additional 100 μl of blocking buffer. 30 μl of a solution of yeastTFIIS (65 μg/ml) was incubated in each of the protease-coated wells for2-4 hours at room temperature. 5 μl of the protein solution was thenmade to 2% Sodium dodecyl sulphate, 25% glycerol, 0.1M Tris-Hel (pH 8.0)and resolved by gel electrophoresis. The results from a typicaldigestion (chymotrypsin) are shown in FIG. 1. The individual proteolyticproducts were purified either by reverse-phase liquid chromatography orby elution from the gel slice and were analyzed by matrix-assisteddesorption time-of-flight mass spectrometry. The fragments correspondedto known domains of the yeast TFIIS (Proc. Nat. Acad. Sci. U.S.A. 93:10604-10608, 1996).

[0085] Stability

[0086] Experiments with plates containing immobilized proteaseslyophilized and stored at 4 degrees celcius for up to one weekdemonstrated results similar to those freshly prepared (data not shown).

[0087] While the present invention has been described with reference towhat are presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0088] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

We claim:
 1. A method for the preparation of proteolytically digestedfragments of a protein, free of protease, for the determination ofdomains and the boundaries of the domains in said protein, the methodcomprises: (1) contacting a quantity of said protein with at least twoconcentrations of a protease under conditions which result in digestedfragments of said protein, wherein each of said concentrations ofprotease is immobilized in a separate compartment; and (2) separatingsaid fragments from said immobilized protease.
 2. A method according toclaim 1 wherein said compartment is in an apparatus which comprises aplurality of compartments.
 3. A method according to claim 2 wherein eachcompartment containing a quantity of said protease contains aconcentration of said protease distinct from the concentration of saidprotease in every other compartment.
 4. A method according to claim 3wherein at least three concentrations of protease are each in one of atleast three different said compartments.
 5. A method according to claim4 wherein said plurality of compartments forms a matrix in a linearformat with at least three increasing concentrations of protease in saidcompartments.
 6. A method according to anyone of claims 1-5 wherein saidmatrix contains more than one protease.
 7. A method according to claim 6wherein said matrix is in a two-dimensional format wherein: (i) distinctconcentrations of each protease are aligned in increasing concentration,along a first axis or concentration axis; and (ii) different proteasesare aligned along a second axis that is perpendicular to the first axis.8. A method according to anyone of claims 1-7 wherein said protease isselected from the group consisting of aminopeptidase M; bromelain;carboxypeptidase A, B and Y; chymopapain; chymotrypsin; clostripain;collagenase; elastase; endoproteinase Arg-C, Glu-C and LysC; Factor Xa;ficin; Gelatinase; kallikrein; metalloendopeptinidase; papain; pepsin;plasmin; plasminogen; peptidase; pronase; proteinase A; proteinase K;subsilisin; thermolysin; thrombin; and trypsin.
 9. A method according toclaim 8 wherein the proteases are trypsin, chymotrypsin, proteinase K orpapain.
 10. A method according to claim 9 wherein the concentrations ofeach of the proteases along the first axis in each case is from about 50μg/mL to about 0.5 μg/mL.
 11. A method according to claim 10 wherein theconcentration for each of the proteases is about 50 μg/mL in onecontainer of a concentration axis; about 25 μg/mL in a second containerof the concentration axis; about 5 μg/mL in a third container of theconcentration axis; about 2.5 μg/mL in a fourth container of theconcentration axis; about 0.5 μg/mL in a fifth container of theconcentration axis; about 0 μg/mL in a sixth container of theconcentration axis.
 12. A method according to any one of claims 1-11wherein the surface upon which said protease is immobilized has beentreated with a blocker of surface interaction.
 13. A method according toclaim 12 wherein the blocker is selected from the group consisting ofBSA and beta-octyl glucoside.
 14. A method according to claim 1-13wherein the apparatus may be prepared and stored prior to use.
 15. Amethod according to any one of claims 1 to 13 wherein the surface towhich said protease is immobilized is selected from the group consistingof an organic support of material selected from the group consisting ofpolyesters, polyamides, polyacrylates, polymethacrylates,polyacrylamides, poly(acrylic acid)m, poly(methacrylic acid);poly(galacturonic acid); poly(aspartic acid); ethylene-maleic anhydridecopolymers; polyolefins; cellulose; cellulose derivatives; agarose gels;dextran gels and derivatives thereof, polysaccharides; polypeptides;collagen; and an inorganic support material selected from the groupconsisting of siliceous and nonsiliceous metal oxides.
 16. A methodaccording to any one of claims 1 to 14 wherein said apparatus is amicrotitre plate.
 17. A method according to claim 16 wherein one of theconditions is time for incubation and is between about 2 to 4 hours. 18.A method according to claim 17 wherein one of the conditions istemperature for a reaction and is room temperature.
 19. A methodaccording to anyone of claims 1 to 18 wherein only limited digestion ofsaid protein occurs.
 20. A method for determining the boundaries of aproteolytically digested fragment of a protein which comprises thesteps: (i) incubating a protein, according to the method of any one ofclaims 1-19 to yield protein fragments; (ii) isolatation of fragment(s)of interest; (iii) determining the mass of the isolated fragment(s); and(iv) matching the mass of the proteolytic fragment(s) to a protein aminoacid sequence of the protein digested.
 21. A method according to claim20 wherein the determination of mass is by a method selected from thegroup consisting of nanospray time-of-flight mass spectrometry andmatrix-assisted time-of-flight mass spectrometry.
 22. A method accordingto claim 21 wherein the protein fragment isolation is carried out by atechnique selected from the group consisting of HPLC and SDS PAGE.
 23. Amethod according to claim 20, 21, or 22 which is automated.
 24. Anapparatus for degradation of a protein, said apparatus comprising aplurality of compartments, each compartment containing a quantity of aprotease immobilized on a surface in each compartment wherein at leasttwo compartments contain different concentrations of said protease. 25.An apparatus according to claim 24 wherein each compartment containing aquantity of said protease contains a concentration of said proteasedistinct from the concentration of said protease in every othercompartment.
 26. An apparatus according to claim 25 wherein at leastthree concentrations of protease are in said compartments.
 27. Anapparatus according to claim 26 wherein said plurality of compartmentsforms a matrix in a linear format with at least three increasingconcentrations of protease in said compartments.
 28. An apparatusaccording to anyone of claims 24-27 wherein said matrix contains morethan one protease.
 29. An apparatus according to claim 28 wherein saidmatrix is in a two-dimensional format wherein: (i) distinctconcentrations of each protease are aligned in increasing concentration,along a first axis or concentration axis; and (ii) different proteasesare aligned along a second axis that is perpendicular to the first axis.30. An apparatus according to anyone of claims 24-29 wherein saidprotease is selected from the group consisting of aminopeptidase M;bromelain; carboxypeptidase A, B and Y; chymopapain; chymotrypsin;clostripain; collagenase; elastase; endoproteinase Arg-C, Glu-C andLysC; Factor Xa; ficin; Gelatinase; kallikrein; metalloendopeptinidase;papain; pepsin; plasmin; plasminogen; peptidase; pronase; proteinase A;proteinase K; subsilisin; thermolysin; thrombin; and trypsin.
 31. Anapparatus according to claim 30 wherein the proteases are trypsin,chymotrypsin, proteinase K or papain.
 32. An apparatus according toclaim 31 wherein the concentrations of each of the proteases along thefirst axis in each case is from about 50 μg/mL to about 0.5 μg/mL. 33.An apparatus according to claim 32 wherein the concentration for each ofthe proteases is about 50 μg/mL in one container of a concentrationaxis; about 25 μg/mL in a second container of the concentration axis;about 5 μg/mL in a third container of the concentration axis; about 2.5μg/mL in a fourth container of the concentration axis; about 0.5 μg/mLin a fifth container of the concentration axis; about 0 μg/mL in a sixthcontainer of the concentration axis.
 34. An apparatus according to anyone of claims 24-33 wherein the surface upon which said protease isimmobilized has been treated with a blocker of surface interaction. 35.An apparatus according to claim 34 wherein the blocker is selected fromthe group consisting of BSA and beta-octyl glucoside.
 36. An apparatusaccording to any one of claims 24 to 33 wherein the surface to whichsaid protease is immobilized is selected from the group consisting of anorganic support of material selected from the group consisting ofpolyesters, polyamides, polyacrylates, polymethacrylates,polyacrylamides, poly(acrylic acid)m, poly(methacrylic acid);poly(galacturonic acid); poly(aspartic acid); ethylene-maleic anhydridecopolymers; polyolefins; cellulose; cellulose derivatives; agarose gels;dextran gels and derivatives thereof, polysaccharides; polypeptides;collagen; and an inorganic support material selected from the groupconsisting of siliceous and nonsiliceous metal oxides.
 37. An apparatusaccording to claim 24-36 wherein the apparatus may be prepared andstored prior to use.
 38. An apparatus according to any one of claims 24to 37 wherein said apparatus is a microtitre plate.